Charyl M. Dutton

Polymerase Chain Reaction Amplification of Specific Alleles: A General Method of Detection of Mutations, Polymorphisms, and Haplotypes

Publisher Summary The polymerase chain reaction (PCR) can be adapted for the rapid detection of known single-base changes in DNA by using specially designed oligonucleotides in a method called “PCR amplification of specific alleles (PASA).” PASA has been further modified to include two allele-specific primers in the same PCR reaction. By varying the lengths of the allele-specific oligonucleotides, the amplified products can be distinguished from each other by gel electrophoresis. This method, called PCR amplification of multiple specific alleles (PAMSA), allows the rapid detection of more than one allele in a single PCR reaction. PASA is a generally applicable technique for the detection of point mutations or polymorphisms. The method may also be used to detect the presence of small deletions or insertions. PASA has the advantages of being rapid, reproducible, nonisotopic, and amenable to automation.

siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line

Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP‐1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP‐1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP‐1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP‐1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP‐1 gene expression and protein synthesis was demonstrated via RT‐PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP‐1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP‐1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP‐1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis.

Inhibition of MMP-1 expression by antisense RNA decreases invasiveness of human chondrosarcoma.

We previously reported that an elevated level of matrix metalloproteinase‐1 (MMP‐1) gene expression in patients with chondrosarcoma has a strong statistical correlation with recurrence and in vitro invasion. In the present study, we used an antisense RNA strategy for MMP‐1 inhibition to determine if this would affect the invasive characteristics of the cells. We transfected a human chondrosarcoma cell line with a retroviral plasmid expressing a 770 bp genomic fragment of the human MMP‐1 gene in the sense or antisense orientation. The results show that cells transfected with the MMP‐1 antisense fragment had a significant decrease in both MMP‐1 protein and enzyme activity (p < 0.05) as compared to cells transfected with an empty plasmid or the parental cells. Cells transfected with the MMP‐1 antisense fragment demonstrated a significant decrease in their ability to invade the collagen I barrier (p < 0.05). The gene expression for MMP‐8 and MMP‐13 were unaffected in cells transfected with the MMP‐1 antisense fragment, MMP‐1 sense fragment, or empty plasmid. These results support the hypothesis that MMP‐1 facilitates tumor cell egress from chondrosarcoma tissue and demonstrate the potential of MMP‐1 as a promising target for a novel biologic therapy in chondrosarcoma.

Cloning of the mouse sodium iodide symporter.

The iodide-concentrating ability of the thyroid gland is essential to the production of thyroid hormone. We report the nucleotide and amino acid sequence of the mouse sodium iodide symporter (mNIS), which mediates this activity within the thyroid gland. An open reading frame of 1,857 nucleotides codes for a protein of 618 amino acids with 95% identity to rat NIS and 84% identity to human NIS. Transient expression of the mNIS cDNA in Chinese hamster ovary (CHO) cells, a nonthyroid cell line, resulted in sodium-dependent, perchlorate-sensitive iodide uptake. Western blot analysis of membrane preparations of CHO cells transiently transfected with mNIS cDNA showed a band of 90 kd when probed with an antibody directed against rat NIS. mNIS will serve as an important reagent in determining the role of NIS in experimental thyroid diseases and for monitoring the immune response to in animal models of NIS-mediated gene therapy.

Absence of a Correlation between the Presence of a Single Nucleotide Polymorphism in the Matrix Metalloproteinase 1 Promoter and Outcome in Patients of Chondrosarcoma

Purpose: Increased levels of matrix metalloproteinase 1 (MMP-1) expression have been associated with poor outcome in chondrosarcoma. The existence of a single nucleotide polymorphism creating an Ets-binding site in the MMP-1 promoter may be one mechanism for elevated MMP-1 transcription. The aim of our study was to identify the prevalence of this single nucleotide polymorphism (SNP) in chondrosarcoma patients, to determine its correlation with disease outcome, and to discern whether it could serve as a prognostic marker in patients with chondrosarcoma. Experimental Design: Sixty-seven chondrosarcoma specimens were selected sequentially from an established tumor bank with a median duration of 47 months follow-up (range, 24 to 179 months). DNA was extracted, amplified with PCR, and sequenced to determine presence (GG) or absence of the Ets-binding site created by the SNP. Results: Eighteen (27%) samples were homozygous for the absence of the Ets site, 34 (51%) were heterozygous for the SNP, and 15 (22%) were homozygous for the SNP. The 5-year overall survival rate for patients was 78, 80, and 84%, respectively (P = 0.5527). The disease-free survival rate was 16, 63, and 76%, respectively (P = 0.0801). The 5-year disease-free survival rate for patients with the homozygous G/G genotype was 16%, compared with 71% for patients who were either homozygous or heterozygous for the GG allele (P = 0.0444). Conclusions: Despite a statistical correlation between MMP-1 gene expression and outcome in chondrosarcoma, this study demonstrates an absence of a correlation between the presence of the SNP and prognosis in patients with chondrosarcoma.

Cryptic dinucleotide polymorphism in the 3' region of the factor IX gene shows substantial variation among different populations.

Long tandem dinucleotide repeats composed of alternating purines and pyrimidines [RY(i)] are abundant and highly polymorphic. “Simple” RY(i) are predominately composed of one tandem repeat of a dinucleotide sequence. In contrast, “cryptic” RY(i [cRY(i)] are composed of multiple short dinucleotide repeats. Herein, we describe the racial distribution of alleles for a polymorphic cRY(i) in the factor IX gene. Allele I is absent in Asians, whereas allele III is rare or absent in Caucasians or blacks. A polymorphic cRY(i) analyzed previously shows even more dramatic variation among racial groups, hinting that a battery of cRY(i) might have utility in assessing the racial origin of a DNA sample.

[45] – Polymerase Chain Reaction Amplification of Specific Alleles: A General Method of Detection of Mutations, Polymorphisms, and Haplotypes

Publisher Summary The polymerase chain reaction (PCR) utilizes two oligonucleotide primers to amplify a segment of DNA more than 1 million-fold. The PCR can be adapted for the rapid detection of known single-base changes in DNA by using specially designed oligonucleotides in PCR amplification of specific alleles (PASA). The principle of this method is to design an oligonucleotide primer that will preferentially amplify one allele over another. PASA is a generally applicable technique for detection of point mutations or polymorphisms and the presence of small deletions or insertion. But PASA has certain disadvantages that are eliminated by another technique, PCR amplification of multiple specific alleles (PAMSA). This technique allows the rapid detection of more than one allele in a single PCR reaction. PAMSA is useful for detecting mutations and polymorphisms. This chapter discusses general method of detecting haplotypes. PASA has been used to perform population screening, haplotype analysis, patient screening, and carrier testing. Haplotypes are useful in population genetics and medicine. However, determining linkage of haplotypes in the absence of DNA samples from appropriate family members can be difficult and laborious. PASA can be adapted to provide a rapid and reproducible method for haplotyping an individual in the absence of relatives. This method, termed “double PASA,” uses four pairs of allele-specific PCR primers to differentially amplify each of the four possible haplotypes from two biallelic polymorphisms. Double PASA is an important tool for haplotyping doubly heterozygous individuals because the physical linkage of alleles on a strand of DNA is necessary to determine the haplotype.